US10697965B2 - Method for treating and prognosing cancer - Google Patents

Method for treating and prognosing cancer Download PDF

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US10697965B2
US10697965B2 US15/554,599 US201615554599A US10697965B2 US 10697965 B2 US10697965 B2 US 10697965B2 US 201615554599 A US201615554599 A US 201615554599A US 10697965 B2 US10697965 B2 US 10697965B2
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cancer
dnmt3a
isgf3γ
peptide
expression level
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Pierre-Francois CARTRON
Mathilde CHERAY
Francois Valette
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Centre National de la Recherche Scientifique CNRS
Universite dAngers
Universite de Nantes
Institut National de la Sante et de la Recherche Medicale INSERM
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Universite dAngers
Universite de Nantes
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/10—Peptides having 12 to 20 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/118—Prognosis of disease development
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
    • G01N2333/4701—Details
    • G01N2333/4703—Regulators; Modulating activity
    • G01N2333/4706—Regulators; Modulating activity stimulating, promoting or activating activity
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/91—Transferases (2.)
    • G01N2333/91005—Transferases (2.) transferring one-carbon groups (2.1)
    • G01N2333/91011—Methyltransferases (general) (2.1.1.)
    • G01N2333/91017—Methyltransferases (general) (2.1.1.) with definite EC number (2.1.1.-)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/04—Screening involving studying the effect of compounds C directly on molecule A (e.g. C are potential ligands for a receptor A, or potential substrates for an enzyme A)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • the present invention relates to an in vitro method for determine the prognosis of the survival time of a patient suffering from a cancer comprising the steps consisting of i) determining the expression level of the couple DNMT3A/ISGF3 ⁇ in a sample from said patient, ii) comparing said expression level with a predetermined reference value and iii) providing a good prognosis when the expression level is lower than the predetermined reference value and a poor prognosis when the expression level is higher than the predetermined reference value.
  • the invention also relates a compound which is a DNMT3A/ISGF3 ⁇ antagonist or a compound which is a DNMT3A/ISGF3 ⁇ gene expression inhibitor for use in the treatment and prevention of cancer.
  • DNA methylation patterns are frequently aberrant in cancer cells. Thus, hypomethylation of intergenic regions can occur, leading to tumorigenesis via the activation of transposable elements and increased genomic instability. Local hypomethylation of genes promoters can promote oncogene expression, while local hypermethylation of the genes promoters can lead to loss of tumor suppressor function in cancer cells. Based on this last point, drug development has focused on DNA methylation inhibitors with the goal of activating tumor suppressor genes (TSG) silenced by DNA methylation. But in absence of specificity, a DNMT inhibitor can promote the demethylation of TSG but also of oncogenes and transposable elements. Thus, the use of unspecific DNMT inhibitors can be anti-tumorigenic or pro-tumorigenic.
  • TSG tumor suppressor genes
  • 5-aza-2′-deoxycytidine treatment an unspecific DNMT inhibitor
  • 5-aza-2′-deoxycytidine treatment increases the invasiveness of non-invasive breast cancer cell lines MCF-7 cells and ZR-75-1 and dramatically induced pro-metastatic genes [Chik F et al., 2011].
  • 5-aza-2′-deoxycytidine treatment is also reported as an inducer of glioma from astrocytes and as an enhancer of tumorigenic property of glioma cells.
  • 5-aza-2′-deoxycytidine is approved by the Food and Drug Administration of the United States for the myelodysplastic syndrome treatment, where it demonstrates significant, although usually transient, improvement in patient survival.
  • DNMT1 can be inhibited by using RG108, MG98 or Procainamide
  • DNMT3A while DNMT3B can be specifically inhibited by using Theaflavin 3, 3′-digallate or NanaomycinA, respectively [Amato R et al., 2012; Kuck D et al., 2010; Kuck D et al., 2010; Lee B et al., 2005 and Rajavelu A et al., 2001].
  • DNMT inhibitors can be also addressed against a specific DNMT/protein-x interaction [Cheray M et al., 2013].
  • D3A-BP DNMT3A-binding protein
  • the present invention relates to an in vitro method for determine the prognosis of the survival time of a patient suffering from a cancer comprising the steps consisting of i) determining the expression level of the couple DNMT3A/ISGF3 ⁇ in a sample from said patient, ii) comparing said expression level with a predetermined reference value and iii) providing a good prognosis when the expression level is lower than the predetermined reference value and a poor prognosis when the expression level is higher than the predetermined reference value.
  • the invention also relates a compound which is a DNMT3A/ISGF3 ⁇ antagonist or a compound which is a DNMT3A/ISGF3 ⁇ gene expression inhibitor for use in the treatment and prevention of cancer.
  • the first aspect of the invention relates to an in vitro method for determining the prognosis of the survival time of a patient suffering from a cancer comprising the steps consisting of i) determining the expression level of the couple DNMT3A/ISGF3 ⁇ in a sample from said patient, ii) comparing said expression level with a predetermined reference value and iii) providing a good prognosis when the expression level is lower than the predetermined reference value and a poor prognosis when the expression level is higher than the predetermined reference value.
  • the invention also relates to an in vitro method for predicting the survival time of a patient suffering from a cancer and treated with conventional treatment comprising the steps consisting of i) determining the expression level of the couple DNMT3A/ISGF3 ⁇ in a sample from said patient, ii) comparing said expression level with a predetermined reference value and iii) providing a good prognosis when the expression level is lower than the predetermined reference value and a poor prognosis when the expression level is higher than the predetermined reference value.
  • the invention also relates to an in vitro method for predicting the response of a patient suffering from a cancer and treated with conventional treatment comprising the steps consisting of i) determining the expression level of the couple DNMT3A/ISGF3 ⁇ in a sample from said patient, ii) comparing said expression level with a predetermined reference value and iii) providing a good response when the expression level is lower than the predetermined reference value and a poor response when the expression level is higher than the predetermined reference value.
  • the terms “conventional treatment” denote any compounds, combination of compounds, combination of chemotherapeutic treatment and radiotherapeutic agent and combination of chemotherapeutic treatment and radiation which may be used for the treatment of cancer.
  • the conventional treatment may the use of a combination of the temozolomide and radiation.
  • the invention also relates to a method for predicting the survival time of a patient suffering from a glioblastoma and treated with radiation and temozolomide comprising the steps consisting of i) determining the expression level of the couple DNMT3A/ISGF3 ⁇ in a sample from said patient, ii) comparing said expression level with a predetermined reference value and iii) providing a good prognosis when the expression level is lower than the predetermined reference value and a poor prognosis when the expression level is higher than the predetermined reference value.
  • the invention also relates to a method for predicting the response of a patient suffering from a glioblastoma and treated with radiation and temozolomide comprising the steps consisting of i) determining the expression level of the couple DNMT3A/ISGF3 ⁇ in a sample from said patient, ii) comparing said expression level with a predetermined reference value and iii) providing a good response when the expression level is lower than the predetermined reference value and a poor response when the expression level is higher than the predetermined reference value.
  • the cancer may be any solid or liquid cancer.
  • the cancer may be selected from the group consisting of bile duct cancer (e.g. periphilar cancer, distal bile duct cancer, intrahepatic bile duct cancer), bladder cancer, bone cancer (e.g. osteoblastoma, osteochrondroma, hemangioma, chondromyxoid fibroma, osteosarcoma, chondrosarcoma, fibrosarcoma, malignant fibrous histiocytoma, giant cell tumor of the bone, chordoma, lymphoma, multiple myeloma), brain and central nervous system cancer (e.g.
  • bile duct cancer e.g. periphilar cancer, distal bile duct cancer, intrahepatic bile duct cancer
  • bladder cancer e.g. osteoblastoma, osteochrondroma, hemangioma, chondromyxoid fibroma, osteosarcom
  • breast cancer e.g. ductal carcinoma in situ, infiltrating ductal carcinoma, infiltrating, lobular carcinoma, lobular carcinoma in, situ, gynecomastia
  • Castleman disease e.g. giant lymph node hyperplasia, angiofollicular lymph node hyperplasia
  • cervical cancer colorectal cancer
  • endometrial cancer e.g.
  • lung cancer e.g. small cell lung cancer, non-small cell lung cancer
  • mesothelioma plasmacytoma, nasal cavity and paranasal sinus cancer (e.g. esthesioneuroblastoma, midline granuloma), nasopharyngeal cancer, neuroblastoma, oral cavity and oropharyngeal cancer, ovarian cancer, pancreatic cancer, penile cancer, pituitary cancer, prostate cancer, retinoblastoma, rhabdomyosarcoma (e.g.
  • rhabdomyosarcoma embryonal rhabdomyosarcoma, alveolar rhabdomyosarcoma, pleomorphic rhabdomyosarcoma), salivary gland cancer, skin cancer (e.g. melanoma, nonmelanoma skin cancer), stomach cancer, testicular cancer (e.g. seminoma, nonseminoma germ cell cancer), thymus cancer, thyroid cancer (e.g. follicular carcinoma, anaplastic carcinoma, poorly differentiated carcinoma, medullary thyroid carcinoma, thyroid lymphoma), vaginal cancer, vulvar cancer, and uterine cancer (e.g. uterine leiomyosarcoma).
  • skin cancer e.g. melanoma, nonmelanoma skin cancer
  • stomach cancer testicular cancer (e.g. seminoma, nonseminoma germ cell cancer), thymus cancer, thyroid cancer (e.g. follicular carcinoma, anaplastic carcinoma
  • the glioblastoma is a glioblastoma multiforme.
  • the sample according to the invention may be a blood, plasma, serum sample or a cancer biopsy.
  • said sample is a glioblastoma biopsy.
  • DNMT3A for “DNA methyltransferase 3A” (Entrez Gene ID number: 1788; mRNA sequence reference: NM_022552.4; protein sequence reference: Q9Y6K1) designed a de novo DNA methyltransferase i.e. an enzyme responsible for the establishment of de novo genomic DNA methylation patterns and, as such, involved in normal development as well as in many diseases including cancer.
  • Protein sequence of DNMT3A (SEQ ID NO:3) is:
  • ISGF3 ⁇ (Entrez Gene ID number: 10379; mRNA sequence reference: NM_006084.4; protein sequence reference: Q00978) denotes a Transcription factor that mediates signaling by type I IFNs.
  • ISGF3 ⁇ binds to the IFN stimulated response element (ISRE) to activate the transcription of interferon stimulated genes, which drive the cell in an antiviral state.
  • ISRE IFN stimulated response element
  • the invention relates to an in vitro method for determine the prognosis of the overall survival (OS) of a patient suffering from a cancer comprising the steps consisting of i) determining the expression level of the couple DNMT3A/ISGF3 ⁇ in a sample from said patient, ii) comparing said expression level with a predetermined reference value and iii) providing a good prognosis when the expression level is lower than the predetermined reference value and a poor prognosis when the expression level is higher than the predetermined reference value.
  • OS overall survival
  • the invention also relates to a method for predicting the overall survival (OS) of a patient suffering from a cancer and treated with conventional treatment comprising the steps consisting of i) determining the expression level of the couple DNMT3A/ISGF3 ⁇ in a sample from said patient, ii) comparing said expression level with a predetermined reference value and iii) providing a good prognosis when the expression level is lower than the predetermined reference value and a poor prognosis when the expression level is higher than the predetermined reference value.
  • OS overall survival
  • the invention also relates to a method for predicting the overall survival (OS) of a patient suffering from a glioblastoma and treated with radiation and temozolomide comprising the steps consisting of i) determining the expression level of the couple DNMT3A/ISGF3 ⁇ in a sample from said patient, ii) comparing said expression level with a predetermined reference value and iii) providing a good prognosis when the expression level is lower than the predetermined reference value and a poor prognosis when the expression level is higher than the predetermined reference value.
  • OS overall survival
  • OS Overall survival
  • the term “Good Prognosis” denotes a patient with more than 50% chance of survival for the next 3 years after the treatment.
  • the invention also relates to a method for predicting the responsiveness of a patient affected with a glioblastoma to a temozolomide and radiation treatment comprising the steps consisting of i) determining the expression level of the couple DNMT3A/ISGF3 ⁇ in a sample from said patient ii) comparing the expression level determined at step i) with its predetermined reference value wherein when the expression level determined at step i) is lower than its predetermined reference values then the responsiveness of the patient to the treatment is good, and when the expression level determined at step i) is higher than its predetermined reference value then the responsiveness of the patient to the treatment is bad.
  • the determination of the expression level of the couple DNMT3A/ISGF3 ⁇ may be determined before or after the beginning of the treatment of the patient.
  • the patient affected with a glioblastoma is mainly treated with a standard treatment consisting of maximal surgical resection, radiotherapy, and concomitant adjuvant chemotherapy with temozolomide.
  • determining the expression level of includes qualitative and/or quantitative detection (measuring levels) with or without reference to a control.
  • expression level of the couple DNMT3A/ISGF3 ⁇ may be measured for example by enzyme-labeled and mediated immunoassays (such as ELISA), flow cytometry assessment or qRT-PCR performed on the sample.
  • the “reference value” may be a healthy subject, i.e. a subject who does not suffer from any cancer and particularly glioblastoma. Particularly, said control is a healthy subject.
  • Detection of the expression level of the couple DNMT3A/ISGF3 ⁇ in the sample may be performed by measuring the level of DNMT3A/ISGF3 ⁇ proteins or the DNMT3A/ISGF3 ⁇ genes.
  • the methods may comprise contacting a sample with a binding partner capable of selectively interacting with DNMT3A/ISGF3 ⁇ proteins present in the sample.
  • the binding partner is generally an antibody that may be polyclonal or monoclonal, particularly monoclonal.
  • the presence of the protein can be detected using standard electrophoretic and immunodiagnostic techniques, including immunoassays such as competition, direct reaction, or sandwich type assays.
  • immunoassays include, but are not limited to, Western blots; agglutination tests; enzyme-labeled and mediated immunoassays, such as ELISAs; biotin/avidin type assays; radioimmunoassays; immunoelectrophoresis; immunoprecipitation, etc.
  • the reactions generally include revealing labels such as fluorescent, chemiluminescent, radioactive, enzymatic labels or dye molecules, or other methods for detecting the formation of a complex between the antigen and the antibody or antibodies reacted therewith.
  • the aforementioned assays generally involve separation of unbound protein in a liquid phase from a solid phase support to which antigen-antibody complexes are bound.
  • Solid supports which can be used in the practice of the invention include substrates such as nitrocellulose (e. g., in membrane or microtiter well form); polyvinylchloride (e. g., sheets or microtiter wells); polystyrene latex (e.g., beads or microtiter plates); polyvinylidine fluoride; diazotized paper; nylon membranes; activated beads, magnetically responsive beads, and the like.
  • an ELISA method can be used, wherein the wells of a microtiter plate are coated with a set of antibodies against the proteins to be tested. A sample containing or suspected of containing the marker protein is then added to the coated wells. After a period of incubation sufficient to allow the formation of antibody-antigen complexes, the plate(s) can be washed to remove unbound moieties and a detectably labeled secondary binding molecule is added. The secondary binding molecule is allowed to react with any captured sample marker protein, the plate is washed and the presence of the secondary binding molecule is detected using methods well known in the art.
  • immunoenzymatic staining methods are known in the art for detecting a protein of interest. For example, immunoenzymatic interactions can be visualized using different enzymes such as peroxidase, alkaline phosphatase, or different chromogens such as DAB, AEC, or Fast Red; or fluorescent labels such as FITC, Cy3, Cy5, Cy7, Alexafluors, etc.
  • Counterstains may include H&E, DAPI, Hoechst, so long as such stains are compatable with other detection reagents and the visualization strategy used.
  • amplification reagents may be used to intensify staining signal.
  • tyramide reagents may be used.
  • the staining methods of the present invention may be accomplished using any suitable method or system as would be apparent to one of skill in the art, including automated, semi-automated or manual systems.
  • the method of the invention may comprise a further step consisting of comparing DNMT3A/ISGF3 ⁇ proteins expression with a control reference.
  • the term “expression level of DNMT3A/ISGF3 ⁇ ” refers to an amount or a concentration of a transcription product, for instance mRNA coding for DNMT3A/ISGF3 ⁇ genes.
  • a level of mRNA expression can be expressed in units such as transcripts per cell or nanograms per microgram of tissue.
  • a level of protein can be expressed as nanograms per microgram of tissue or nanograms per milliliter of a culture medium, for example.
  • relative units can be employed to describe an expression level.
  • Measuring the expression level of a gene can be performed by a variety of techniques well known in the art.
  • the expression level of a gene may be determined by determining the quantity of mRNA.
  • Methods for determining the quantity of mRNA are well known in the art.
  • the nucleic acid contained in the samples e.g., cell or tissue prepared from the patient
  • the extracted mRNA is then detected by hybridization (e. g., Northern blot analysis, in situ hybridization) and/or amplification (e.g., RT-PCR).
  • LCR ligase chain reaction
  • TMA transcription-mediated amplification
  • SDA strand displacement amplification
  • NASBA nucleic acid sequence based amplification
  • Nucleic acids having at least 10 nucleotides and exhibiting sequence complementarity or homology to the mRNA of interest herein find utility as hybridization probes or amplification primers. It is understood that such nucleic acids need not be identical, but are typically at least about 80% identical to the homologous region of comparable size, more particularly 85% identical and even more particularly 90-95% identical. In certain embodiments, it will be advantageous to use nucleic acids in combination with appropriate means, such as a detectable label, for detecting hybridization.
  • the nucleic acid probes include one or more labels, for example to permit detection of a target nucleic acid molecule using the disclosed probes.
  • a nucleic acid probe includes a label (e.g., a detectable label).
  • a “detectable label” is a molecule or material that can be used to produce a detectable signal that indicates the presence or concentration of the probe (particularly the bound or hybridized probe) in a sample.
  • a labeled nucleic acid molecule provides an indicator of the presence or concentration of a target nucleic acid sequence (e.g., genomic target nucleic acid sequence) (to which the labeled uniquely specific nucleic acid molecule is bound or hybridized) in a sample.
  • a label associated with one or more nucleic acid molecules can be detected either directly or indirectly.
  • a label can be detected by any known or yet to be discovered mechanism including absorption, emission and/or scattering of a photon (including radio frequency, microwave frequency, infrared frequency, visible frequency and ultra-violet frequency photons).
  • Detectable labels include colored, fluorescent, phosphorescent and luminescent molecules and materials, catalysts (such as enzymes) that convert one substance into another substance to provide a detectable difference (such as by converting a colorless substance into a colored substance or vice versa, or by producing a precipitate or increasing sample turbidity), haptens that can be detected by antibody binding interactions, and paramagnetic and magnetic molecules or materials.
  • detectable labels include fluorescent molecules (or fluorochromes).
  • fluorescent molecules or fluorochromes
  • Numerous fluorochromes are known to those of skill in the art, and can be selected, for example from Life Technologies (formerly Invitrogen), e.g., see, The Handbook—A Guide to Fluorescent Probes and Labeling Technologies).
  • fluorophores that can be attached (for example, chemically conjugated) to a nucleic acid molecule (such as a uniquely specific binding region) are provided in U.S. Pat. No.
  • fluorophores include thiol-reactive europium chelates which emit at approximately 617 mn (Heyduk and Heyduk, Analyt. Biochem. 248:216-27, 1997; J. Biol. Chem. 274:3315-22, 1999), as well as GFP, LissamineTM, diethylaminocoumarin, fluorescein chlorotriazinyl, naphthofluorescein, 4,7-dichlororhodamine and xanthene (as described in U.S. Pat. No. 5,800,996 to Lee et al.) and derivatives thereof.
  • fluorophores known to those skilled in the art can also be used, for example those available from Life Technologies (Invitrogen; Molecular Probes (Eugene, Oreg.)) and including the ALEXA FLUOR® series of dyes (for example, as described in U.S. Pat. Nos. 5,696,157, 6,130,101 and 6,716,979), the BODIPY series of dyes (dipyrrometheneboron difluoride dyes, for example as described in U.S. Pat. Nos.
  • a fluorescent label can be a fluorescent nanoparticle, such as a semiconductor nanocrystal, e.g., a QUANTUM DOTTM (obtained, for example, from Life Technologies (QuantumDot Corp, Invitrogen Nanocrystal Technologies, Eugene, Oreg.); see also, U.S. Pat. Nos. 6,815,064; 6,682,596; and 6,649, 138).
  • Semiconductor nanocrystals are microscopic particles having size-dependent optical and/or electrical properties.
  • semiconductor nanocrystals When semiconductor nanocrystals are illuminated with a primary energy source, a secondary emission of energy occurs of a frequency that corresponds to the handgap of the semiconductor material used in the semiconductor nanocrystal. This emission can be detected as colored light of a specific wavelength or fluorescence.
  • Semiconductor nanocrystals with different spectral characteristics are described in e.g., U.S. Pat. No. 6,602,671.
  • semiconductor nanocrystals can be produced that are identifiable based on their different spectral characteristics.
  • semiconductor nanocrystals can be produced that emit light of different colors based on their composition, size or size and composition.
  • quantum dots that emit light at different wavelengths based on size (565 mn, 655 mn, 705 mn, or 800 mn emission wavelengths), which are suitable as fluorescent labels in the probes disclosed herein are available from Life Technologies (Carlshad, Calif.).
  • Additional labels include, for example, radioisotopes (such as 3H), metal chelates such as DOTA and DPTA chelates of radioactive or paramagnetic metal ions like Gd3+, and liposomes.
  • radioisotopes such as 3H
  • metal chelates such as DOTA and DPTA chelates of radioactive or paramagnetic metal ions like Gd3+
  • liposomes include, for example, radioisotopes (such as 3H), metal chelates such as DOTA and DPTA chelates of radioactive or paramagnetic metal ions like Gd3+, and liposomes.
  • Detectable labels that can be used with nucleic acid molecules also include enzymes, for example horseradish peroxidase, alkaline phosphatase, acid phosphatase, glucose oxidase, beta-galactosidase, beta-glucuronidase, or beta-lactamase.
  • enzymes for example horseradish peroxidase, alkaline phosphatase, acid phosphatase, glucose oxidase, beta-galactosidase, beta-glucuronidase, or beta-lactamase.
  • an enzyme can be used in a metallographic detection scheme.
  • SISH silver in situ hyhridization
  • Metallographic detection methods include using an enzyme, such as alkaline phosphatase, in combination with a water-soluble metal ion and a redox-inactive substrate of the enzyme. The substrate is converted to a redox-active agent by the enzyme, and the redoxactive agent reduces the metal ion, causing it to form a detectable precipitate.
  • Metallographic detection methods also include using an oxido-reductase enzyme (such as horseradish peroxidase) along with a water soluble metal ion, an oxidizing agent and a reducing agent, again to form a detectable precipitate.
  • an oxido-reductase enzyme such as horseradish peroxidase
  • Probes made using the disclosed methods can be used for nucleic acid detection, such as ISH procedures (for example, fluorescence in situ hybridization (FISH), chromogenic in situ hybridization (CISH) and silver in situ hybridization (SISH)) or comparative genomic hybridization (CGH).
  • ISH procedures for example, fluorescence in situ hybridization (FISH), chromogenic in situ hybridization (CISH) and silver in situ hybridization (SISH)
  • CGH comparative genomic hybridization
  • ISH In situ hybridization
  • a sample containing target nucleic acid sequence e.g., genomic target nucleic acid sequence
  • a metaphase or interphase chromosome preparation such as a cell or tissue sample mounted on a slide
  • a labeled probe specifically hybridizable or specific for the target nucleic acid sequence (e.g., genomic target nucleic acid sequence).
  • the slides are optionally pretreated, e.g., to remove paraffin or other materials that can interfere with uniform hybridization.
  • the sample and the probe are both treated, for example by heating to denature the double stranded nucleic acids.
  • the probe (formulated in a suitable hybridization buffer) and the sample are combined, under conditions and for sufficient time to permit hybridization to occur (typically to reach equilibrium).
  • the chromosome preparation is washed to remove excess probe, and detection of specific labeling of the chromosome target is performed using standard techniques.
  • a biotinylated probe can be detected using fluorescein-labeled avidin or avidin-alkaline phosphatase.
  • fluorescein-labeled avidin or avidin-alkaline phosphatase For fluorochrome detection, the fluorochrome can be detected directly, or the samples can be incubated, for example, with fluorescein isothiocyanate (FITC)-conjugated avidin. Amplification of the FITC signal can be effected, if necessary, by incubation with biotin-conjugated goat antiavidin antibodies, washing and a second incubation with FITC-conjugated avidin.
  • FITC fluorescein isothiocyanate
  • samples can be incubated, for example, with streptavidin, washed, incubated with biotin-conjugated alkaline phosphatase, washed again and pre-equilibrated (e.g., in alkaline phosphatase (AP) buffer).
  • AP alkaline phosphatase
  • Numerous reagents and detection schemes can be employed in conjunction with FISH, CISH, and SISH procedures to improve sensitivity, resolution, or other desirable properties.
  • probes labeled with fluorophores including fluorescent dyes and QUANTUM DOTS®
  • fluorophores including fluorescent dyes and QUANTUM DOTS®
  • the probe can be labeled with a nonfluorescent molecule, such as a hapten (such as the following non-limiting examples: biotin, digoxigenin, DNP, and various oxazoles, pyrrazoles, thiazoles, nitroaryls, benzofurazans, triterpenes, ureas, thioureas, rotenones, coumarin, courmarin-based compounds, Podophyllotoxin, Podophyllotoxin-based compounds, and combinations thereof), ligand or other indirectly detectable moiety.
  • a hapten such as the following non-limiting examples: biotin, digoxigenin, DNP, and various oxazoles, pyrrazoles, thiazoles, nitroaryls, benzofurazans, triterpenes, ureas, thioureas, rotenones, coumarin, courmarin-based compounds, Podophyllotoxin, Podo
  • Probes labeled with such non-fluorescent molecules (and the target nucleic acid sequences to which they bind) can then be detected by contacting the sample (e.g., the cell or tissue sample to which the probe is bound) with a labeled detection reagent, such as an antibody (or receptor, or other specific binding partner) specific for the chosen hapten or ligand.
  • a labeled detection reagent such as an antibody (or receptor, or other specific binding partner) specific for the chosen hapten or ligand.
  • the detection reagent can be labeled with a fluorophore (e.g., QUANTUM DOT®) or with another indirectly detectable moiety, or can be contacted with one or more additional specific binding agents (e.g., secondary or specific antibodies), which can be labeled with a fluorophore.
  • the probe, or specific binding agent (such as an antibody, e.g., a primary antibody, receptor or other binding agent) is labeled with an enzyme that is capable of converting a fluorogenic or chromogenic composition into a detectable fluorescent, colored or otherwise detectable signal (e.g., as in deposition of detectable metal particles in SISH).
  • the enzyme can be attached directly or indirectly via a linker to the relevant probe or detection reagent. Examples of suitable reagents (e.g., binding reagents) and chemistries (e.g., linker and attachment chemistries) are described in U.S. Patent Application Publication Nos. 2006/0246524; 2006/0246523, and 2007/01 17153.
  • multiplex detection schemes can be produced to facilitate detection of multiple target nucleic acid sequences (e.g., genomic target nucleic acid sequences) in a single assay (e.g., on a single cell or tissue sample or on more than one cell or tissue sample).
  • a first probe that corresponds to a first target sequence can be labelled with a first hapten, such as biotin, while a second probe that corresponds to a second target sequence can be labelled with a second hapten, such as DNP.
  • the bound probes can be detected by contacting the sample with a first specific binding agent (in this case avidin labelled with a first fluorophore, for example, a first spectrally distinct QUANTUM DOT®, e.g., that emits at 585 mn) and a second specific binding agent (in this case an anti-DNP antibody, or antibody fragment, labelled with a second fluorophore (for example, a second spectrally distinct QUANTUM DOT®, e.g., that emits at 705 mn).
  • a first specific binding agent in this case avidin labelled with a first fluorophore, for example, a first spectrally distinct QUANTUM DOT®, e.g., that emits at 585 mn
  • a second specific binding agent in this case an anti-DNP antibody, or antibody fragment, labelled with a second fluorophore (for example, a second spectrally distinct QUANTUM DOT®,
  • Probes typically comprise single-stranded nucleic acids of between 10 to 1000 nucleotides in length, for instance of between 10 and 800, more particularly of between 15 and 700, typically of between 20 and 500.
  • Primers typically are shorter single-stranded nucleic acids, of between 10 to 25 nucleotides in length, designed to perfectly or almost perfectly match a nucleic acid of interest, to be amplified.
  • the probes and primers are “specific” to the nucleic acids they hybridize to, i.e. they particularly hybridize under high stringency hybridization conditions (corresponding to the highest melting temperature Tm, e.g., 50% formamide, 5 ⁇ or 6 ⁇ SCC. SCC is a 0.15 M NaCl, 0.015 M Na-citrate).
  • the nucleic acid primers or probes used in the above amplification and detection method may be assembled as a kit.
  • a kit includes consensus primers and molecular probes.
  • a particular kit also includes the components necessary to determine if amplification has occurred.
  • the kit may also include, for example, PCR buffers and enzymes; positive control sequences, reaction control primers; and instructions for amplifying and detecting the specific sequences.
  • the methods of the invention comprise the steps of providing total RNAs extracted from cumulus cells and subjecting the RNAs to amplification and hybridization to specific probes, more particularly by means of a quantitative or semi-quantitative RT-PCR.
  • the expression level is determined by DNA chip analysis.
  • DNA chip or nucleic acid microarray consists of different nucleic acid probes that are chemically attached to a substrate, which can be a microchip, a glass slide or a microsphere-sized bead.
  • a microchip may be constituted of polymers, plastics, resins, polysaccharides, silica or silica-based materials, carbon, metals, inorganic glasses, or nitrocellulose.
  • Probes comprise nucleic acids such as cDNAs or oligonucleotides that may be about 10 to about 60 base pairs.
  • a sample from a test subject optionally first subjected to a reverse transcription, is labelled and contacted with the microarray in hybridization conditions, leading to the formation of complexes between target nucleic acids that are complementary to probe sequences attached to the microarray surface.
  • the labelled hybridized complexes are then detected and can be quantified or semi-quantified. Labelling may be achieved by various methods, e.g. by using radioactive or fluorescent labelling.
  • Many variants of the microarray hybridization technology are available to the man skilled in the art (see e.g. the review by Hoheisel, Nature Reviews, Genetics, 2006, 7:200-210).
  • Expression level of a gene may be expressed as absolute expression level or normalized expression level.
  • expression levels are normalized by correcting the absolute expression level of a gene by comparing its expression to the expression of a gene that is not a relevant for determining the cancer stage of the patient, e.g., a housekeeping gene that is constitutively expressed.
  • Suitable genes for normalization include housekeeping genes such as the actin gene ACTB, ribosomal 18S gene, GUSB, PGK1 and TFRC. According to the invention the housekeeping genes used were GAPDH, GUSB, TBP and ABL1. This normalization allows the comparison of the expression level in one sample, e.g., a patient sample, to another sample, or between samples from different sources.
  • a “threshold value”, “threshold level”, “reference value” or “cut-off value” can be determined experimentally, empirically, or theoretically.
  • a threshold value can also be arbitrarily selected based upon the existing experimental and/or clinical conditions, as would be recognized by a person of ordinary skilled in the art. Particularly, the person skilled in the art may compare the expression levels of the couple DNMT3A/ISGF3 ⁇ obtained according to the method of the invention with a defined threshold value.
  • said threshold value is the mean expression level of the couple DNMT3A/ISGF3 ⁇ of a population of healthy individuals.
  • the term “healthy individual” denotes a human which is known to be healthy, i.e. which does not suffer from a cancer and in particular from a glioblastoma and does not need any medical care.
  • the skilled person in the art may determine the expression level of the couple DNMT3A/ISGF3 ⁇ in a biological sample, particularly a biopsy of a glioblastoma cancer for example, of 100 individuals known to be healthy.
  • the mean value of the obtained expression levels is then determined, according to well known statistical analysis, so as to obtain the mean expression level of the couple DNMT3A/ISGF3 ⁇ . Said value is then considered as being normal and thus constitutes a threshold value.
  • the physician is then able to classify and prognostic the cancer.
  • the physician would be able to adapt and optimize appropriate medical care of a subject in a critical and life-threatening condition suffering from cancer.
  • the determination of said prognosis is highly appropriate for follow-up care and clinical decision making.
  • kits useful for the methods of the invention comprising means for detecting DNMT3A/ISGF3 ⁇ expression.
  • kits of the invention may comprise an anti-DNMT3A protein antibody and an anti-ISGF3 ⁇ ; and another molecule coupled with a signalling system which binds to said DNMT3A/ISGF3 ⁇ antibodies or any molecule which bind to the mRNA of DNMT3A/ISGF3 ⁇ genes like a probe.
  • the antibodies or combination of antibodies are in the form of solutions ready for use.
  • the kit comprises containers with the solutions ready for use. Any other forms are encompassed by the present invention and the man skilled in the art can routinely adapt the form to the use in immunohistochemistry.
  • the invention relates to an in vitro method for monitoring a patient's response cancer treatment which comprises a step of measuring the expression level of the couple DNMT3A/ISGF3 ⁇ , in a sample from a patient.
  • the present invention relates to the use of the couple DNMT3A/ISGF3 ⁇ as a biomarker for the monitoring of anti-cancer therapies and more particularly an anti-glioblastoma therapy.
  • Another aspect of the invention relates to a compound which is an antagonist of the couple DNMT3A/ISGF3 ⁇ or an inhibitor of the expression of the couple DNMT3A/ISGF3 ⁇ for use in the treatment of patient suffering of a cancer with a high expression level of the couple DNMT3A/ISGF3 ⁇ .
  • the invention also relates to a compound which is an antagonist of the couple DNMT3A/ISGF3 ⁇ or an inhibitor of the expression of the couple DNMT3A/ISGF3 ⁇ for use in the treatment of patient suffering from a glioblastoma with a high expression level of the couple DNMT3A/ISGF3 ⁇ .
  • a second aspect of the invention relates to a compound which is a DNMT3A/ISGF3 ⁇ antagonist or a compound which is a DNMT3A/ISGF3 ⁇ gene expression inhibitor for use in the treatment and prevention of cancer.
  • DNMT3A/ISGF3 ⁇ antagonist is meant a natural or synthetic compound that has a biological effect opposite to that of a natural ligand of DNMT3A and/or ISGF3 ⁇ .
  • the antagonist binds to the couple DNMT3A/ISGF3 ⁇ and blocks the action of these proteins by competing with the ligand of these proteins.
  • An antagonist is defined by its ability to block the actions of a natural ligand.
  • DNMT3A/ISGF3 ⁇ antagonist refers to any DNMT3A/ISGF3 ⁇ antagonist that is currently known in the art or that will be identified in the future, and includes any chemical entity that, upon administration to a subject, results in inhibition of a biological activity associated with activation of the DNMT3A/ISGF3 ⁇ proteins in the subject, including any of the downstream biological effects otherwise resulting from the binding to DNMT3A/ISGF3 ⁇ of its natural ligands.
  • an antagonist can act by occupying the ligand binding site or a portion thereof of DNMT3A/ISGF3 ⁇ proteins, thereby making the receptor inaccessible to its natural ligand so that its normal biological activity is prevented or reduced.
  • the antagonist of the invention can block the binding of the protein DNMT3A to the protein ISGF3 ⁇ .
  • a “DNMT3A/ISGF3 ⁇ antagonist” can be a compound which binds to the DNMT3A protein or to the ISGF3 ⁇ protein.
  • a DNMT3A/ISGF3 ⁇ antagonist may bind to the DNMT3A protein at the position 85-99, 103-129, 178-207, 235-246, 256-273, 331-360, 409-433 or 547-574 of SEQ ID NO: 3 and inhibits the DNMT3A/ISGF3 ⁇ interaction.
  • the present invention also relates to a method of screening a candidate compound for use as a drug for the prevention and treatment of cancer in a subject in need thereof, wherein the method comprises the steps of: i) providing candidate compounds and ii) selecting candidate compounds that block or antagonise DNMT3A/ISGF3 ⁇ .
  • the present invention relates to a method of screening a candidate compound for use as a drug for the treatment and prevention of cancer in a subject in need thereof, wherein the method comprises the steps of:
  • a test may be used. For example, a test using bioluminescence resonance energy transfer (BRET) system for assaying DNMT3A/ISGF3 interactions and identifying molecule having the ability to inhibit this interaction can be develop.
  • BRET Bioluminescence Resonance Energy Transfer
  • cDNA of interDNMT3A and ISGF3g will be inserted in vectors designed for Bioluminescence Resonance Energy Transfer (BRET) experiments (pEYFP and phRluc, Invitrogen). Next, these vectors will be transfected in U251 cells (a GBM cell lines).
  • BRET Bioluminescence Resonance Energy Transfer
  • DNMT3A/ISGF3 ⁇ gene expression inhibitor is meant a natural or synthetic compound which inhibits the expression of the DNMT3A gene expression or the ISGF3 ⁇ gene expression or both.
  • a test may be used.
  • qPCR and ELISA experiments can be performed.
  • the invention also relates to i) compound according to the invention, and ii) a chemotherapeutic agent, as a combined preparation for simultaneous, separate or sequential for use in the treatment and prevention of cancer.
  • the invention also relates to i) compound according to the invention, and ii) a chemotherapeutic agent and iii) a radiotherapy or a radiotherapeutic agent, as a combined preparation for simultaneous, separate or sequential for use in the treatment and prevention of cancer.
  • radiotherapy may consist of gamma-radiation, X-ray radiation, electrons or photons, external radiotherapy or curitherapy.
  • the term “radiotherapeutic agent”, is intended to refer to any radiotherapeutic agent known to one of skill in the art to be effective to treat or ameliorate cancer, without limitation.
  • the radiotherapeutic agent can be an agent such as those administered in brachytherapy or radionuclide therapy.
  • Such methods can optionally further comprise the administration of one or more additional cancer therapies, such as, but not limited to, chemotherapies, and/or another radiotherapy.
  • the chemotherapeutic agent may be the temozolomide, 5-aza-2′-deoxycytidine, Theaflavin 3, 3′-digallate, zebularine, decitabine, 4-amino-N-(4-aminophenyl), benzamide analogues of quinoline-based SGI-1027 (PMID: 24678024 or 23294304.
  • the cancer according to the invention is a glioblastoma.
  • the invention relates to i) compound according to the invention, and ii) a chemotherapeutic agent and iii) a radiotherapy, as a combined preparation for simultaneous, separate or sequential for use in the treatment and prevention of glioblastoma.
  • the invention relates to i) compound according to the invention, and ii) the temozolomide and iii) a radiotherapy, as a combined preparation for simultaneous, separate or sequential for use in the treatment and prevention of glioblastoma.
  • the compound according to the invention includes but is not limited to a small organic molecule, an antibody, an intra-antibody, a nanobody and a polypeptide.
  • the compound according to the invention may be a low molecular weight compound, e. g. a small organic molecule (natural or not).
  • small organic molecule refers to a molecule (natural or not) of a size comparable to those organic molecules generally used in pharmaceuticals.
  • particular small organic molecules range in size up to about 10000 Da, more particularly up to 5000 Da, more particularly up to 2000 Da and most particularly up to about 1000 Da.
  • the compound according to the invention is an antibody, an intra-antibody or a nanobody.
  • Antibodies, intra-antibodies or nanobodies directed against DNMT3A or ISGF3 ⁇ proteins can be raised according to known methods by administering the appropriate antigen or epitope to a host animal selected, e.g., from pigs, cows, horses, rabbits, goats, sheep, and mice, among others.
  • a host animal selected, e.g., from pigs, cows, horses, rabbits, goats, sheep, and mice, among others.
  • Various adjuvants known in the art can be used to enhance antibody production.
  • antibodies useful in practicing the invention can be polyclonal or monoclonal antibodies.
  • Monoclonal antibodies against DNMT3A or ISGF3 ⁇ proteins can be prepared and isolated using any technique that provides for the production of antibody molecules by continuous cell lines in culture.
  • Techniques for production and isolation include but are not limited to the hybridoma technique originally described by Kohler and Milstein (1975); the human B-cell hybridoma technique (Cote et al., 1983); and the EBV-hybridoma technique (Cole et al. 1985).
  • techniques described for the production of single chain antibodies can be adapted to produce anti-DNMT3A or anti-ISGF3 ⁇ proteins single chain antibodies.
  • Anti-DNMT3A or anti-ISGF3 ⁇ antibody fragments including but not limited to F(ab′)2 fragments, which can be generated by pepsin digestion of an intact antibody molecule, and Fab fragments, which can be generated by reducing the disulfide bridges of the F(ab′)2 fragments.
  • F(ab′)2 fragments which can be generated by pepsin digestion of an intact antibody molecule
  • Fab fragments which can be generated by reducing the disulfide bridges of the F(ab′)2 fragments.
  • Fab and/or scFv expression libraries can be constructed to allow rapid identification of fragments having the desired specificity to DNMT3A or ISGF3 ⁇ proteins.
  • Humanized anti-DNMT3A or anti-ISGF3 ⁇ antibodies and antibody fragments therefrom can also be prepared according to known techniques.
  • “Humanized antibodies” are forms of non-human (e.g., rodent) chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin.
  • humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region (CDRs) of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity and capacity.
  • donor antibody such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity and capacity.
  • framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues.
  • humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance.
  • the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence.
  • the humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.
  • Fc immunoglobulin constant region
  • neutralizing antibodies of DNMT3A or ISGF3 ⁇ are selected.
  • the compound according to the invention is an anti-DNMT3A antibody.
  • the antibody according to the invention may be the ab23565 antibody bought by Abcam or the H-295 antibody bought by Santa Cruz.
  • the compound according to the invention is an anti-ISGF3 ⁇ antibody.
  • the compound according to the invention is an aptamer.
  • Aptamers are a class of molecule that represents an alternative to antibodies in term of molecular recognition.
  • Aptamers are oligonucleotide or oligopeptide sequences with the capacity to recognize virtually any class of target molecules with high affinity and specificity.
  • Such ligands may be isolated through Systematic Evolution of Ligands by EXponential enrichment (SELEX) of a random sequence library, as described in Tuerk C. and Gold L., 1990.
  • the random sequence library is obtainable by combinatorial chemical synthesis of DNA. In this library, each member is a linear oligomer, eventually chemically modified, of a unique sequence.
  • Peptide aptamers consists of a conformationally constrained antibody variable region displayed by a platform protein, such as E. coli Thioredoxin A that are selected from combinatorial libraries by two hybrid methods (Colas et al., 1996).
  • neutralizing aptamers of DNMT3A or ISGF3 ⁇ are selected.
  • the compound according to the invention is a peptide, a polypeptide or a protein.
  • the peptide, the polypeptide or the protein can be a functional equivalent of DNMT3A or ISGF3 ⁇ .
  • a “functional equivalent” of DNMT3A or ISGF3 ⁇ is a compound which is capable of binding to DNMT3A or ISGF3 ⁇ .
  • the term “functional equivalent” or “function-conservative variants” include fragments, mutants, and muteins of DNMT3A or ISGF3 ⁇ .
  • the term “functionally equivalent” thus includes any equivalent of DNMT3A or ISGF3 ⁇ obtained by altering the amino acid sequence, for example by one or more amino acid deletions, substitutions or additions. Amino acid substitutions may be made, for example, by point mutation of the DNA encoding the amino acid sequence.
  • the functional equivalents include soluble forms of DNMT3A or ISGF3 ⁇ .
  • a suitable soluble form of these proteins, or functional equivalents thereof, might comprise, for example, a truncated form of the protein from which the transmembrane domain has been removed by chemical, proteolytic or recombinant methods.
  • the functional equivalent is at least 80% homologous to the corresponding protein.
  • the functional equivalent is at least 90% homologous as assessed by any conventional analysis algorithm such as for example, the Pileup sequence analysis software (Program Manual for the Wisconsin Package, 1996).
  • a functionally equivalent fragment as used herein also may mean any fragment or assembly of fragments of DNMT3A or ISGF3 ⁇ .
  • Functionally equivalent fragments may belong to the same protein family as the DNMT3A or ISGF3 ⁇ identified herein.
  • protein family is meant a group of proteins that share a common function and exhibit common sequence homology.
  • homology between functionally equivalent protein sequences is at least 25% across the whole of amino acid sequence of the complete protein. More particularly, the homology is at least 50%, even more particularly 75% across the whole of amino acid sequence of the protein or protein fragment. More particularly, homology is greater than 80% across the whole of the sequence. More particularly, homology is greater than 90% across the whole of the sequence. More particularly, homology is greater than 95% across the whole of the sequence.
  • polypeptides of the invention may be produced by any suitable means, as will be apparent to those of skill in the art.
  • expression may conveniently be achieved by culturing under appropriate conditions recombinant host cells containing the polypeptide of the invention.
  • the polypeptide is produced by recombinant means, by expression from an encoding nucleic acid molecule.
  • Systems for cloning and expression of a polypeptide in a variety of different host cells are well known.
  • the polypeptide When expressed in recombinant form, the polypeptide may be generated by expression from an encoding nucleic acid in a host cell.
  • a host cell Any host cell may be used, depending upon the individual requirements of a particular system. Suitable host cells include bacteria mammalian cells, plant cells, yeast and baculovirus systems. Mammalian cell lines available in the art for expression of a heterologous polypeptide include Chinese hamster ovary cells. HeLa cells, baby hamster kidney cells and many others. Bacteria are also hosts for the production of recombinant protein, due to the ease with which bacteria may be manipulated and grown. A common bacterial host is E. coli.
  • polypeptides used in the therapeutic methods of the present invention may be modified in order to improve their therapeutic efficacy.
  • modification of therapeutic compounds may be used to decrease toxicity, increase circulatory time, or modify biodistribution.
  • the toxicity of potentially important therapeutic compounds can be decreased significantly by combination with a variety of drug carrier vehicles that modify biodistribution.
  • adding dipeptides can improve the penetration of a circulating agent in the eye through the blood retinal barrier by using endogenous transporters.
  • a strategy for improving drug viability is the utilization of water-soluble polymers.
  • Various water-soluble polymers have been shown to modify biodistribution, improve the mode of cellular uptake, change the permeability through physiological barriers; and modify the rate of clearance from the body.
  • water-soluble polymers have been synthesized that contain drug moieties as terminal groups, as part of the backbone, or as pendent groups on the polymer chain.
  • PEG Polyethylene glycol
  • Attachment to various drugs, proteins, and liposomes has been shown to improve residence time and decrease toxicity.
  • PEG can be coupled to active agents through the hydroxyl groups at the ends of the chain and via other chemical methods; however, PEG itself is limited to at most two active agents per molecule.
  • copolymers of PEG and amino acids were explored as novel biomaterials which would retain the biocompatibility properties of PEG, but which would have the added advantage of numerous attachment points per molecule (providing greater drug loading), and which could be synthetically designed to suit a variety of applications.
  • PEGylation techniques for the effective modification of drugs.
  • drug delivery polymers that consist of alternating polymers of PEG and tri-functional monomers such as lysine have been used by VectraMed (Plainsboro, N.J.).
  • the PEG chains typically 2000 daltons or less
  • Such copolymers retain the desirable properties of PEG, while providing reactive pendent groups (the carboxylic acid groups of lysine) at strictly controlled and predetermined intervals along the polymer chain.
  • the reactive pendent groups can be used for derivatization, cross-linking, or conjugation with other molecules.
  • These polymers are useful in producing stable, long-circulating pro-drugs by varying the molecular weight of the polymer, the molecular weight of the PEG segments, and the cleavable linkage between the drug and the polymer.
  • the molecular weight of the PEG segments affects the spacing of the drug/linking group complex and the amount of drug per molecular weight of conjugate (smaller PEG segments provides greater drug loading).
  • increasing the overall molecular weight of the block co-polymer conjugate will increase the circulatory half-life of the conjugate. Nevertheless, the conjugate must either be readily degradable or have a molecular weight below the threshold-limiting glomular filtration (e.g., less than 60 kDa).
  • linkers may be used to maintain the therapeutic agent in a pro-drug form until released from the backbone polymer by a specific trigger, typically enzyme activity in the targeted tissue.
  • a specific trigger typically enzyme activity in the targeted tissue.
  • tissue activated drug delivery is particularly useful where delivery to a specific site of biodistribution is required and the therapeutic agent is released at or near the site of pathology.
  • Linking group libraries for use in activated drug delivery are known to those of skill in the art and may be based on enzyme kinetics, prevalence of active enzyme, and cleavage specificity of the selected disease-specific enzymes. Such linkers may be used in modifying the protein or fragment of the protein described herein for therapeutic delivery.
  • the peptide of the invention is the peptide P1 (SEQ ID NO:1).
  • the invention also relates to a peptide comprising the amino acids sequence: RPMPRLTFQAGDPYYI (SEQ ID NO:1) or a function-conservative variant thereof.
  • the peptide comprising the amino acids sequence SEQ ID NO: 1 or a function-conservative variant may be used for treating or preventing cancer.
  • the invention relates to i) a compound which is the peptide P1 (SEQ ID NO:1) or a function-conservative variant thereof, and ii) the temozolomide, as a combined preparation for simultaneous, separate or sequential for use in the treatment and prevention of cancer.
  • the invention relates to i) a compound which is the peptide P1 (SEQ ID NO:1) or a function-conservative variant thereof, and ii) the temozolomide, as a combined preparation for simultaneous, separate or sequential for use in the treatment and prevention of glioblastoma.
  • the invention relates to i) a compound which is the peptide P1 (SEQ ID NO:1), and ii) the temozolomide and iii) a radiotherapy, as a combined preparation for simultaneous, separate or sequential for use in the treatment and prevention of cancer.
  • the invention relates to i) a compound which is the peptide P1 (SEQ ID NO:1), and ii) the temozolomide and iii) a radiotherapy, as a combined preparation for simultaneous, separate or sequential for use in the treatment and prevention of glioblastoma.
  • the compound is a functionally equivalent fragment of the peptide P1.
  • the peptide P1 of SEQ ID NO:1 is used to sensitive cancer cell to a chemotherapeutic agent and particularly to temozolomide.
  • the invention relates to a peptide comprising the amino acids sequence SEQ ID NO:1 or a function-conservative variant thereof.
  • the invention also encompasses peptides that are function-conservative variants of the peptide comprising SEQ ID NO: 1 as described here above.
  • the peptide according to the invention may differ from 1, 2 or 3 amino acids to the SEQ ID NO:1.
  • the peptide according to the invention may differ from 4 or 5 amino acids to the SEQ ID NO:1.
  • the peptide of the invention comprises at least 75% identity over said the SEQ ID NO: 1, even more preferably at least 80%, at least 85%, at least 90%, at least 95%, at least 97% and is still able to decrease tumor cell proliferation or still able to induce PCD in tumor cell.
  • the peptide of the invention consists in the amino acid sequence as set forth in SEQ ID NO:1 or a variant thereof comprising at least 75%, preferably at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identity with SEQ ID NO:1 and is still able for disrupting the DNMT3A/ISGF3 ⁇ interaction.
  • said peptide is an amino acid sequence of less than 50 amino acids long that comprises the amino acid sequence SEQ ID NO:1 as defined here above.
  • said soluble peptide is an amino acid sequence of less than 45 amino acids long that comprises the amino acid sequence SEQ ID NO:1 as defined here above.
  • said soluble peptide is an amino acid sequence of less than 40 amino acids long that comprises the amino acid sequence SEQ ID NO:1 as defined here above.
  • said soluble peptide is an amino acid sequence of less than 30 amino acids long that comprises the amino acid sequence SEQ ID NO:1 as defined here above.
  • said soluble peptide is an amino acid sequence of less than 20 amino acids long that comprises the amino acid sequence SEQ ID NO:1 as defined here above.
  • said soluble peptide is an amino acid sequence of less than 15 amino acids long that comprises the amino acid sequence SEQ ID NO:1 as defined here above.
  • the peptide, the polypeptide or the protein of the invention and particularly the peptide P1 is linked with at least one cell penetrating peptide (CPP).
  • CPP cell penetrating peptide
  • cell penetrating peptide or “CPP” are used interchangeably and refer to cationic cell penetrating peptides, also called transport peptides, carrier peptides, or peptide transduction domains.
  • CPP as shown herein, have the capability of inducing cell penetration of a peptide fused to the CPP within 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of cells of a given cell culture population, including all integers in between, and allow macromolecular translocation within multiple tissues in vivo upon systemic administration.
  • a cell-penetrating peptide may also refer to a peptide which, when brought into contact with a cell under appropriate conditions, passes from the external environment in the intracellular environment, including the cytoplasm, organelles such as mitochondria, or the nucleus of the cell, in conditions significantly greater than passive diffusion.
  • penetrating peptides may be those described in Fonseca S. B. et al., Advanced Drug Delivery Reviews, 2009, 61: 953-964, Johansson et al., Methods in Molecular Biology, 2011, Vol.
  • the cell penetrating peptide comprises or consists of: Tat peptide, polyarginines peptide, HA2-R9 peptide, Penetratin peptide, Transportan peptide, Vectocell® peptide, maurocalcine peptide, decalysine peptide, HIV-Tat derived PTD4 peptide, Hepatitis B virus Translocation Motif (PTM) peptide, mPrP1-28 peptide, POD, pVEC, EB1, Rath, CADY, Histatin 5, Antp peptide, Cyt86-101 peptide, DPT peptide.
  • Tat peptide polyarginines peptide
  • HA2-R9 peptide Penetratin peptide
  • Transportan peptide Vectocell® peptide
  • maurocalcine peptide decalysine peptide
  • HIV-Tat derived PTD4 peptide HIV-Tat derived PTD4 peptide
  • the peptide, the polypeptide or the protein of the invention is linked to two, three or more penetrating peptides.
  • the compound according to the invention is an inhibitor of DNMT3A or ISGF3 ⁇ gene expression.
  • Small inhibitory RNAs can also function as inhibitors of DNMT3A or ISGF3 ⁇ expression for use in the present invention.
  • DNMT3A or ISGF3 ⁇ gene expression can be reduced by contacting a subject or cell with a small double stranded RNA (dsRNA), or a vector or construct causing the production of a small double stranded RNA, such that DNMT3A or ISGF3 ⁇ gene expression is specifically inhibited (i.e. RNA interference or RNAi).
  • dsRNA small double stranded RNA
  • RNAi RNA interference
  • Methods for selecting an appropriate dsRNA or dsRNA-encoding vector are well known in the art for genes whose sequence is known (e.g. see for example Tuschl, T. et al. (1999); Elbashir, S. M. et al. (2001); Hannon, G J. (2002); McManus, M T. et al.
  • miRNA can be used to as inhibitors of DNMT3A or ISGF3 ⁇ gene expression.
  • miRNA-29a and b, miRNA-143, miRNA-101 and miRNA 369 can be used to inhibit DNMT3A gene expression and miRNA-106 can be used to inhibit ISGF3G gene expression.
  • Ribozymes can also function as inhibitors of DNMT3A or ISGF3 ⁇ gene expression for use in the present invention.
  • Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA.
  • the mechanism of ribozyme action involves sequence specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage.
  • Engineered hairpin or hammerhead motif ribozyme molecules that specifically and efficiently catalyze endonucleolytic cleavage of DNMT3A or ISGF3 ⁇ mRNA sequences are thereby useful within the scope of the present invention.
  • ribozyme cleavage sites within any potential RNA target are initially identified by scanning the target molecule for ribozyme cleavage sites, which typically include the following sequences, GUA, GUU, and GUC. Once identified, short RNA sequences of between about 15 and 20 ribonucleotides corresponding to the region of the target gene containing the cleavage site can be evaluated for predicted structural features, such as secondary structure, that can render the oligonucleotide sequence unsuitable. The suitability of candidate targets can also be evaluated by testing their accessibility to hybridization with complementary oligonucleotides, using, e.g., ribonuclease protection assays.
  • antisense oligonucleotides and ribozymes useful as inhibitors of DNMT3A or ISGF3 ⁇ gene expression can be prepared by known methods. These include techniques for chemical synthesis such as, e.g., by solid phase phosphoramadite chemical synthesis. Alternatively, anti-sense RNA molecules can be generated by in vitro or in vivo transcription of DNA sequences encoding the RNA molecule. Such DNA sequences can be incorporated into a wide variety of vectors that incorporate suitable RNA polymerase promoters such as the T7 or SP6 polymerase promoters. Various modifications to the oligonucleotides of the invention can be introduced as a means of increasing intracellular stability and half-life.
  • Possible modifications include but are not limited to the addition of flanking sequences of ribonucleotides or deoxyribonucleotides to the 5′ and/or 3′ ends of the molecule, or the use of phosphorothioate or 2′-O-methyl rather than phosphodiesterase linkages within the oligonucleotide backbone.
  • Antisense oligonucleotides siRNAs and ribozymes of the invention may be delivered in vivo alone or in association with a vector.
  • a “vector” is any vehicle capable of facilitating the transfer of the antisense oligonucleotide siRNA or ribozyme nucleic acid to the cells and particularly cells expressing DNMT3A or ISGF3 ⁇ .
  • the vector transports the nucleic acid to cells with reduced degradation relative to the extent of degradation that would result in the absence of the vector.
  • the vectors useful in the invention include, but are not limited to, plasmids, phagemids, viruses, other vehicles derived from viral or bacterial sources that have been manipulated by the insertion or incorporation of the the antisense oligonucleotide siRNA or ribozyme nucleic acid sequences.
  • Viral vectors are a particular type of vector and include, but are not limited to nucleic acid sequences from the following viruses: retrovirus, such as moloney murine leukemia virus, harvey murine sarcoma virus, murine mammary tumor virus, and rouse sarcoma virus; adenovirus, adeno-associated virus; SV40-type viruses; polyoma viruses; Epstein-Barr viruses; papilloma viruses; herpes virus; vaccinia virus; polio virus; and RNA virus such as a retrovirus.
  • retrovirus such as moloney murine leukemia virus, harvey murine sarcoma virus, murine mammary tumor virus, and rouse sarcoma virus
  • adenovirus adeno-associated virus
  • SV40-type viruses polyoma viruses
  • Epstein-Barr viruses Epstein-Barr viruses
  • papilloma viruses herpes virus
  • Non-cytopathic viral vectors are based on non-cytopathic eukaryotic viruses in which non-essential genes have been replaced with the gene of interest.
  • Non-cytopathic viruses include retroviruses (e.g., lentivirus), the life cycle of which involves reverse transcription of genomic viral RNA into DNA with subsequent proviral integration into host cellular DNA.
  • Retroviruses have been approved for human gene therapy trials. Most useful are those retroviruses that are replication-deficient (i.e., capable of directing synthesis of the desired proteins, but incapable of manufacturing an infectious particle).
  • retroviral expression vectors have general utility for the high-efficiency transduction of genes in vivo.
  • viruses for certain applications are the adeno-viruses and adeno-associated viruses, which are double-stranded DNA viruses that have already been approved for human use in gene therapy.
  • the adeno-associated virus can be engineered to be replication deficient and is capable of infecting a wide range of cell types and species. It further has advantages such as, heat and lipid solvent stability; high transduction frequencies in cells of diverse lineages, including hemopoietic cells; and lack of superinfection inhibition thus allowing multiple series of transductions.
  • the adeno-associated virus can integrate into human cellular DNA in a site-specific manner, thereby minimizing the possibility of insertional mutagenesis and variability of inserted gene expression characteristic of retroviral infection.
  • adeno-associated virus infections have been followed in tissue culture for greater than 100 passages in the absence of selective pressure, implying that the adeno-associated virus genomic integration is a relatively stable event.
  • the adeno-associated virus can also function in an extrachromosomal fashion.
  • Plasmid vectors have been extensively described in the art and are well known to those of skill in the art. See e.g. Sambrook et al., 1989. In the last few years, plasmid vectors have been used as DNA vaccines for delivering antigen-encoding genes to cells in vivo. They are particularly advantageous for this because they do not have the same safety concerns as with many of the viral vectors. These plasmids, however, having a promoter compatible with the host cell, can express a peptide from a gene operatively encoded within the plasmid.
  • Plasmids may be delivered by a variety of parenteral, mucosal and topical routes.
  • the DNA plasmid can be injected by intramuscular, eye, intradermal, subcutaneous, or other routes. It may also be administered by intranasal sprays or drops, rectal suppository and orally.
  • the plasmids may be given in an aqueous solution, dried onto gold particles or in association with another DNA delivery system including but not limited to liposomes, dendrimers, cochleate and microencapsulation.
  • the antisense oligonucleotide, siRNA, shRNA or ribozyme nucleic acid sequence is under the control of a heterologous regulatory region, e.g., a heterologous promoter.
  • the promoter may be specific for Muller glial cells, microglia cells, endothelial cells, pericyte cells and astrocytes
  • a specific expression in Muller glial cells may be obtained through the promoter of the glutamine synthetase gene is suitable.
  • the promoter can also be, e.g., a viral promoter, such as CMV promoter or any synthetic promoters.
  • Another object of the invention relates to a method for treating and preventing cancer comprising administrating to a subject in need thereof a therapeutically effective amount of a compound which is a DNMT3A/ISGF3 ⁇ antagonist or a compound which is a DNMT3A/ISGF3 ⁇ gene expression inhibitor.
  • the invention relates to a method for treating and preventing glioblastoma comprising administrating to a subject in need thereof a therapeutically effective amount of a compound which is a DNMT3A/ISGF3 ⁇ antagonist or a compound which is a DNMT3A/ISGF3 ⁇ gene expression inhibitor.
  • Another object of the invention relates to a therapeutic composition
  • a therapeutic composition comprising a compound according to the invention for use in the treatment and prevention of cancer.
  • the invention relates to a therapeutic composition
  • a therapeutic composition comprising a compound according to the invention for use in the treatment and prevention of glioblastoma
  • Any therapeutic agent of the invention may be combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form therapeutic compositions.
  • “Pharmaceutically” or “pharmaceutically acceptable” refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate.
  • a pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.
  • compositions for example, the route of administration, the dosage and the regimen naturally depend upon the condition to be treated, the severity of the illness, the age, weight, and sex of the patient, etc.
  • compositions of the invention can be formulated for a topical, oral, intranasal, parenteral, intraocular, intravenous, intramuscular or subcutaneous administration and the like.
  • the pharmaceutical compositions contain vehicles which are pharmaceutically acceptable for a formulation capable of being injected.
  • vehicles which are pharmaceutically acceptable for a formulation capable of being injected.
  • These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions.
  • the doses used for the administration can be adapted as a function of various parameters, and in particular as a function of the mode of administration used, of the relevant pathology, or alternatively of the desired duration of treatment.
  • compositions include, e.g. tablets or other solids for oral administration; time release capsules; and any other form currently can be used.
  • compositions of the present invention may comprise a further therapeutic active agent.
  • the present invention also relates to a kit comprising a compound according to the invention and a further therapeutic active agent.
  • said therapeutic active agent may be an anti-cancer agent.
  • FIG. 1 A high level of DNMT3A/ISGF3 ⁇ interaction is a poor prognosis factor.
  • Kaplan-Meier curves illustrate the difference of overall survival (OS) between patient with high (H) and low (L) levels of DNMT3A/ISGF3 ⁇ interaction.
  • p value is obtained by performing a Cox Proportional Hazards Survival Regression test.
  • FIG. 2 Specific disruption of DNMT3a/ISGF3 ⁇ interaction.
  • a and B Impact of peptides miming the DNMT3A/ISGF3 ⁇ binding regions on the DNMT3A/ISGF3 ⁇ interaction.
  • Pictures and graphs are representatives of three independent pull-down experiments. I: input. p values were obtained by performing a t test.
  • FIG. 3 Effect of a treatment associating the P1 peptide with TMZ in a swiss nude mice model of established tumors.
  • PCTC Primary Cultured Tumor Cells
  • the primary cultured tumor cells were obtained after mechanical dissociation according to the technique previously described. Briefly, tumor tissue was cut into pieces of 1-5 mm3 and plated in a 60 mm2 tissue culture dish with DMEM with 10% FBS and antibiotics. Additionally and in parallel, minced pieces of tumor were incubated with 200 U/ml collagenase I (Sigma, France) and 500 U/ml DNaseI (Sigma, France) in PBS during 1 hr at 37° C. with vigorous constant agitation. The single-cell suspension was filtered through a 70 mm cell strainer (BD Falcon, France), washed with PBS, and suspended in DMEM-10% FBS. Cell cultures were subsequently split 1:2 when confluent and experiments were done before passage 3-5.
  • P-LISA Proximity Ligation In situ Assay
  • Preparations were mounted by using ProLong Gold antifade reagent with DAPI (Life Technologies, France). Pictures acquisition was realized in structured illumination microscopy. After decovolving (3.5 Huygens Essential software (SVI)), 3D view was obtained by using Amira.4.1.1 program. Finally, the images were analyzed by using the freeware “BlobFinder” available for download from www.cb.uu.se/ ⁇ amin/BlobFinder. Thus, we obtained either number of signals per nuclei since nuclei can be automatically identified.
  • Peptides were spotted on an Amino-PEG500-UC540 membrane using a MultiPep peptide synthesizer (Intavis AG, Cologne, Germany) at a loading capacity of 400 nmol/cm2. After synthesis the membrane was dried then the capped side-chains were deprotected by cleavage for 1 h with a cocktail containing 95% trifluoroacetic acid, 3% tri-isopropyl, 2% H2O. The trifluoroacetic acid was removed and the membrane rinsed with dichloromethane, followed by dimethylformaldehyde and then ethanol. The membrane was saturated before incubation with the considered recombinant protein for 2 h at room temperature.
  • a MultiPep peptide synthesizer Intavis AG, Cologne, Germany
  • Pull-down assays were performed by using the GST/His Tagged-Protein Interaction Pull-Down Kits (Thermo Scientific, France). Briefly, 100 ⁇ g of bait protein were immobilized on column via an incubation at 4° C. for 1 h with gentle mixing. After washing, 1 ⁇ g of prey protein was added for 1 h at 4° C. with gentle rocking motion on a rotating platform. After washes and elution, the “bait-prey” interaction was analyzed by SDS-PAGE and Western blot methods. Competitive pull-down experimentations were realized by pre-incubating considered peptides for 1 h at 37° C.
  • proteins were size fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Proteins were transferred onto nitrocellulose or PVDF membrane. Saturation and blotting were realized by using SNAP i.TM Protein Detection System (Millipore, France). The detection of proteins was performed using ECLTM (Amersham Biosciences, France) and/or SuperSignal west femto Maximum Sensitivity (Thermo Scientific, France) chemilumenscence reagents. The detection of proteins was performed using the FusionX7 Imager (Fisher Scientific, France).
  • NLS sequence was added to peptides.
  • Cells were harvested during the exponential growth phase by trypsinization and were resuspended in their original media. They were washed in PBS, pH 7.2 (0.14 M NaCl, 2 mM KCl, 8 mM Na2HPO4 and 1.5 mM NaH2PO4) and resuspended at a concentration of 0.6 ⁇ 106 cells/ml in original culture medium.
  • 0.8 ml of the cell suspension was mixed with the peptides (50 ⁇ g/ml), allowed to stand at room temperature for 10 min and added to a disposable 0.4 cm Bio-Rad electroporation cuvette (Bio-Rad, France).
  • Electroporation efficiency for each cell line was initially determined by flow cytometry by uptake of the fluorescent dye, lucifer yellow (Sigma, France). Electroporation was carried out in a Gene-Pulser (Bio-Rad, France) with cells exposed to one pulse. The following parameters were used: cuvette gap 0.4 cm, voltage 0.3 kV, time constant 35 ms, and capacitor 960 ⁇ F. Following electroporation, cells were allowed to recover by standing at room temperature for 10 min, then removed from the electroporation chamber, washed twice in PBS and resuspended in 2 ml of original culture medium.
  • DNA was extracted by using the QiaAmp DNA mini Kit (Qiagen, France). Next, global DNA methylation was estimated by quantifying the presence of 5-methylcytosine using Methylamp Global DNA methylation Quantification kit (Euromedex-Epigentek, France) according to the manufacturers's instructions.
  • Percentages of cell death were evaluated by using a Trypan Blue Stain 0.4%, and the Countess® Automated Cell Counter (Life Technologies, France). Cell death was induced using temozolomide (25 ⁇ M) and irradtiation (2 Gy) such as previously described.
  • Doubling time i.e. the period of time required for a quantity to double in size
  • Doubling Time Online Calculator website http://www.doubling-time.com/compute.php
  • Cell number was determined, every 24 h over 120 h, using the Countess® Automated Cell Counter (Life Technologies, France).
  • Cell migration assay was performed using a scratch technique.
  • Cells were plated in 6-well plates at 80-90%, and were treated with 10 ⁇ g/ml mitomycin C (Sigma, Farnce) for 2 hours (in order to remove the influence of cell proliferation). Cells were then scratched. Cell migration was monitored by microscopy. The images acquired for each sample were analyzed quantitatively. For each image, distances between one side of scratch and the other were measured. By comparing the images from time 0 to the last time point (24 hours), we obtain the distance of each scratch closure on the basis of the distances that are measured.
  • PCTCs primary cultured tumor cells
  • a High Level of DNMT3A/ISGF3 ⁇ Interaction is a Poor Prognosis Factor.
  • the 31 patients were divided into two groups based on the DNMT3A/ISGF3 ⁇ interaction levels found on their tumor biopsies. Tumors from 15 patients expressed high levels of DNMT3A/ISGF3 ⁇ interaction (higher than the median of DNMT3A/ISGF3 ⁇ interaction, 12.5), while 16 patients had a DNMT3A/ISGF3 ⁇ interaction equal to or lower than 12.5.
  • the positive peptides for an interaction with GST-ISGF3 ⁇ were then detected by using Thyphoon and antibodies directed against ISGF3 ⁇ (data not shown). After fluorescence quantification, the sequences of amino acids of DNMT3A interacting with GST-ISGF3 ⁇ were determined (data not shown). Thus, we observed that the sequences 85-99, 103-129, 178-207, 235-246, 256-273, 331-360, 409-433 and 547-574 of the DNMT3A protein sequence were implicated in the DNMT3A/ISGF3 ⁇ interaction.
  • P-LISA Proximity Ligation In situ Assays
  • P1 was designed to inhibit the DNMT3A/ISGF3 ⁇ interactions. However, P1 could also affect other interaction existing between DNMT3 and a D3A-BP. To investigate this point, we analyzed the effect of P1 on the DNMT3A/D3A-BP interactions of interest. We noted that P1 has no effect on the integrity of the DNMT3A/GATA1, DNMT3A/AP2 ⁇ and DNMT3A/HDAC1 interactions in PCTC #1.
  • TMZ treatment was inefficient to limit tumor growth since no statistical difference was observed between untreated mice and mice treated with TMZ only, and between untrated mice and mice treated with P1 ( FIG. 3B ). More interestingly, we noted that the TMZ+P1 treatment reduced tumors volumes, while the TMZ+P1′ treatment is inefficient to reduce tumor growth. Thus, our data indicated that the use of P1 with TMZ promoted the TMZ-induced reduction of tumor growth.
  • P1 peptide does not promote global DNA hypomethylation and MGMT demethylation.
  • MGMT methylation is associated with a good responsive of anti-glioma treatment including TMZ and irradiation [Esteller M et al., 2000 and Hegi M et al., 2005].
  • TMZ and irradiation Esteller M et al., 2000 and Hegi M et al., 2005.

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